Tamer Al Kayal

Effect of Platelet Lysate on Human Cells Involved in Different Phases of Wound Healing

Background Platelets are rich in mediators able to positively affect cell activity in wound healing. Aim of this study was to characterize the effect of different concentrations of human pooled allogeneic platelet lysate on human cells involved in the different phases of wound healing (inflammatory phase, angiogenesis, extracellular matrix secretion and epithelialization). Methodology/Principal Findings Platelet lysate effect was studied on endothelial cells, monocytes, fibroblasts and keratinocytes, in terms of viability and proliferation, migration, angiogenesis, tissue repair pathway activation (ERK1/2) and inflammatory response evaluation (NFκB). Results were compared both with basal medium and with a positive control containing serum and growth factors. Platelet lysate induced viability and proliferation at the highest concentrations tested (10% and 20% v/v). Whereas both platelet lysate concentrations increased cell migration, only 20% platelet lysate was able to significantly promote angiogenic activity (p<0.05 vs. control), comparably to the positive control. Both platelet lysate concentrations activated important inflammatory pathways such as ERK1/2 and NFκB with the same early kinetics, whereas the effect was different for later time-points. Conclusion/Significance These data suggest the possibility of using allogeneic platelet lysate as both an alternative to growth factors commonly used for cell culture and as a tool for clinical regenerative application for wound healing.

Lysozyme interaction with negatively charged lipid bilayers: protein aggregation and membrane fusion

We report on the interaction of hen egg-white lysozyme (HEWL) with lipid vesicles in terms of surface-induced protein conformational variation and subsequent aggregation. In particular, we investigated the variations of the secondary structure of native lysozyme in the presence of liposomes with different surface charge density, resulting from different molar ratios of the zwitterionic POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and the negatively charged POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)). It is well known that the main driving force involved in the interaction between globally anionic liposomes and lysozyme is electrostatic compensation, which, in some cases, produces extended aggregation. Moreover the presence of membranes can induce unfolding in the protein. In order to understand the main determinants of such phenomena, we probed simultaneously lysozyme-induced vesicle fusion events, variations in the secondary structure of the protein and its effect on liposomal membrane fluidity. We found that above a charge-density threshold, the association with vesicles results in modifications of the native structure associated with a decrease of liposomal membrane fluidity. Electron microscopy images revealed that the above described interactions result in mesoscopic structural changes, i.e. liposome clustering and fusion, together with the appearance of elongated structures, reminiscent of fibrillar aggregates. Additionally, a confocal microscopy analysis revealed that upon interaction with giant unilamellar vesicles (GUVs) of the same lipid composition where the above interactions were observed, a prompt insertion of lysozyme in the membrane occurs, leading to vesicle clustering, with the appearance of elongated structures where both the lipid and the protein are present.

Evaluation of the Effect of a Gamma Irradiated DBM-Pluronic F127 Composite on Bone Regeneration in Wistar Rat

Demineralized bone matrix (DBM) is widely used for bone regeneration. Since DBM is prepared in powder form its handling properties are not optimal and limit the clinical use of this material. Various synthetic and biological carriers have been used to enhance the DBM handling. In this study we evaluated the effect of gamma irradiation on the physical-chemical properties of Pluronic and on bone morphogenetic proteins (BMPs) amount in DBM samples. In vivo studies were carried out to investigate the effect on bone regeneration of a gamma irradiated DBM-Pluronic F127 (DBM-PF127) composite implanted in the femur of rats. Gamma irradiation effects (25 kGy) on physical-chemical properties of Pluronic F127 were investigated by rheological and infrared analysis. The BMP-2/BMP-7 amount after DBM irradiation was evaluated by ELISA. Bone regeneration capacity of DBM-PF127 containing 40% (w/w) of DBM was investigated in transcortical holes created in the femoral diaphysis of Wistar rat. Bone porosity, repaired bone volume and tissue organization were evaluated at 15, 30 and 90 days by Micro-CT and histological analysis. The results showed that gamma irradiation did not induce significant modification on physical-chemical properties of Pluronic, while a decrease in BMP-2/BMP-7 amount was evidenced in sterilized DBM. Micro-CT and histological evaluation at day 15 post-implantation revealed an interconnected trabeculae network in medullar cavity and cellular infiltration and vascularization of DBM-PF127 residue. In contrast a large rate of not connected trabeculae was observed in Pluronic filled and unfilled defects. At 30 and 90 days the DBM-PF127 samples shown comparable results in term of density and thickness of the new formed tissue respect to unfilled defect. In conclusion a gamma irradiated DBM-PF127 composite, although it may have undergone a significant decrease in the concentration of BMPs, was able to maintains bone regeneration capability.

Design and characterization of a composite material based on Sr(II)-loaded clay nanotubes included within a biopolymer matrix

This paper reports on the preparation, characterization, and cytotoxicity of a hybrid nanocomposite material made of Sr(II)-loaded Halloysite nanotubes included within a biopolymer (3-polyhydroxybutyrate-co-3-hydroxyvalerate) matrix. The Sr(II)-loaded inorganic scaffold is intended to provide mechanical resistance, multi-scale porosity, and to favor the in-situ regeneration of bone tissue thanks to its biocompatibility and bioactivity. The interaction of the hybrid system with the physiological environment is mediated by the biopolymer coating, which acts as a binder, as well as a diffusional barrier to the Sr(II) release. The degradation of the polymer progressively leads to the exposure of the Sr(II)-loaded Halloysite scaffold, tuning its interaction with osteogenic cells. The in vitro biocompatibility of the composite was demonstrated by cytotoxicity tests on L929 fibroblast cells. The results indicate that this composite material could be of interest for multiple strategies in the field of bone tissue engineering.

The effect of gamma irradiation on physical–mechanical properties and cytotoxicity of polyurethane–polydimethylsiloxane microfibrillar vascular grafts

Poly(ether) urethane (PEtU)–polydimethylsiloxane (PDMS) based materials have been processed by a spray, phase-inversion technique to produce microfibrillar small-diameter vascular grafts; however the effect of sterilization upon these grafts is still unknown. This study investigated the effect of gamma irradiation on grafts made of PEtU–PDMS materials containing different PDMS concentrations. Sterilisation-induced changes in surface chemical structure and morphology were assessed by infrared spectroscopy, light and scanning electron microscopy. Tensile tests were used to examine changes in mechanical properties and the cytotoxicity evaluation was performed on L929 fibroblasts. The study demonstrated that physical–chemical and mechanical properties of PEtU–PDMS grafts, at each PDMS concentration, were not significantly affected by the exposure to gamma irradiation, moreover no sign of cytotoxicity was observed after sterilisation. Although in vitro experiments have been promising, further in vivo studies are necessary to evaluate the biodegradation behaviour of PEtU–PDMS graft after gamma irradiation, before any clinical application.

Oligonucleotide biofunctionalization enhances endothelial progenitor cell adhesion on cobalt/chromium stents.

As the endothelium still represents the ideal surface for cardiovascular devices, different endothelialization strategies have been attempted for biocompatibility and nonthrombogenicity enhancement. Since endothelial progenitor cells (EPCs) could accelerate endothelialization, preventing thrombosis and restenosis, the aim of this study was to use oligonucleotides (ONs) to biofunctionalize stents for EPC binding. In order to optimize the functionalization procedure before its application to cobalt-chromium (Co/Cr) stents, discs of the same material were preliminarily used. Surface aminosilanization was assessed by infrared spectroscopy and scanning electron microscopy. A fluorescent endothelial-specific ON was immobilized on aminosilanized surfaces and its presence was visualized by confocal microscopy. Fluorescent ON binding to porcine blood EPCs was assessed by flow cytometry. Viability assay was performed on EPCs cultured on unmodified, nontargeting ON or specific ON-coated discs; fluorescent staining of nuclei and F-actin was then performed on EPCs cultured on unmodified or specific ON-coated discs and stents. Disc biofunctionalization significantly increased EPC viability as compared to both unmodified and nontargeting ON-coated surfaces; cell adhesion was also significantly increased. Stents were successfully functionalized with the specific ON, and EPC binding was confirmed by confocal microscopy. In conclusion, stent biofunctionalization for EPC binding was successfully achieved in vitro, suggesting its use to obtain in vivo endothelialization, exploiting the natural regenerative potential of the human body.

Healing effect of a fibrin-based scaffold loaded with platelet lysate in full-thickness skin wounds:

Chronic skin lesions are difficult to heal due to reduced levels and activity of endogenous growth factors. The platelet lysate, obtained by repeated freeze–thawing of platelet-enriched blood samples, is an easily attainable source of a wide range of growth factors and bioactive mediators involved in tissue repair. In this study, a bio-synthetic scaffold composed of poly(ether)urethane–polydimethylsiloxane material and fibrin was developed for platelet lysate delivery to chronic skin wounds. The kinetics release and the bioactivity of growth factors released from platelet lysate–loaded poly(ether)urethane–polydimethylsiloxane–fibrin scaffold were investigated, respectively, by enzyme-linked immunosorbent assay and a cell proliferation test using human fibroblasts. The in vitro experiments demonstrated that the platelet lysate–loaded poly(ether)urethane–polydimethylsiloxane–fibrin scaffold provides a sustained release of platelet derived growth factors. The cell growth in the presence of scaffold was comparable to those observed for the platelet lysate added to culture medium in free form, showing that the scaffold preparation process did not affect biological activity of growth factors. The effect of platelet lysate–loaded poly(ether)urethane–polydimethylsiloxane–fibrin scaffold on wound healing in genetically diabetic mouse (db/db) was also investigated. The application of the scaffold on full-thickness skin wounds significantly accelerated wound closure at day 15 post-surgery compared with control poly(ether)urethane–polydimethylsiloxane–fibrin scaffold (without platelet lysate) or a commercially available polyurethane film dressing. Histological analysis demonstrated an increased re-epithelialization, granulation tissue formation, and collagen deposition. The ability of the platelet lysate–loaded poly(ether)urethane–polydimethylsiloxane–fibrin scaffold to promote wound healing in vivo through simultaneous delivery of multiple active substances suggests its potential use for the treatment of diabetic foot ulcers.

In vivo evaluation of an elastomeric small-diameter vascular graft reinforced with a highly flexible Nitinol mesh: IN VIVO EVALUATION OF REINFORCED VASCULAR GRAFTS

Highly porous small-diameter vascular grafts (SDVGs) prepared with elastomeric materials such as poly(ether urethane) (PEtU)-polydimethylsiloxane (PEtU-PDMS) are capable to biodegrade but may develop aneurismal dilatation. Through a compliance/patency assessment with ultrasound techniques, the current study investigated the functionality, in terms of patency and endothelialization, of a highly flexible and porous Nitinol mesh incorporated into PEtU-PDMS SDVGs in a sheep carotid model. Nitinol-PEtU-PDMS grafts with an internal diameter (ID) of 4 mm were manufactured by spray, phase-inversion technique. Compliance tests were performed by ultrasound (US) imaging using a high-resolution ultrasound diagnostic system. Ten adult sheep were implanted with 7 cm long grafts. The results of this study demonstrated an almost complete neointima luminal coverage in transmurally porous grafts reinforced with the Nitinol meshes after 6 months of implantation. Additionally, ultrasound has been used to quantitatively assess and monitor hemodynamic variables in an experimental model of synthetic vascular graft replacement. The use of reinforced PEtU-PDMS grafts may accelerate the endothelialization process of relatively long grafts, such as those needed for aortocoronary bypass.